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Rab8a Vesicles Regulate Wnt Ligand Delivery and Paneth Cell Maturation

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Rab8a Vesicles Regulate Wnt Ligand Delivery and Paneth Cell Maturation © 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 2147-2162 doi:10.1242/dev.121046 RESEARCH ARTICLE STEM CELLS AND REGENERATION Rab8a vesicles regulate Wnt ligand delivery and Paneth cell maturation at the intestinal stem cell niche Soumyashree Das1, Shiyan Yu1, Ryotaro Sakamori1, Pavan Vedula1, Qiang Feng1, Juan Flores1, Andrew Hoffman2, Jiang Fu3, Ewa Stypulkowski1, Alexis Rodriguez1, Radek Dobrowolski1, Akihiro Harada4, Wei Hsu3, Edward M. Bonder1, Michael P. Verzi2,5 and Nan Gao1,5,* ABSTRACT the cytoplasmic protein Dishevelled (Dvl, or Dsh) (Wong et al., Communication between stem and niche supporting cells maintains 2003), in association with low-density lipoprotein receptor-related the homeostasis of adult tissues. Wnt signaling is a crucial regulator protein 5 and 6 (Lrp5/6), triggering the assembly of a multiprotein of the stem cell niche, but the mechanism that governs Wnt complex at the plasma membrane (Bilic et al., 2007). This plasma membrane-localized protein aggregate, which is sometimes referred ligand delivery in this compartment has not been fully investigated. ‘ ’ We identified that Wnt secretion is partly dependent on Rab8a- to as the Wnt signalosome (Bilic et al., 2007), inactivates a mediated anterograde transport of Gpr177 (wntless), a Wnt-specific cytoplasmic destruction machinery consisting of casein kinase 1, transmembrane transporter. Gpr177 binds to Rab8a, depletion of glycogen synthase kinase 3 (Gsk3), axis inhibitor (Axin), adenomatosis polyposis coli and the E3 ubiquitin ligase β-Trcp which compromises Gpr177 traffic, thereby weakening the secretion β of multiple Wnts. Analyses of generic Wnt/β-catenin targets in Rab8a (Btrc), causing -catenin stabilization (Cadigan and Peifer, 2009; knockout mouse intestinal crypts indicate reduced signaling activities; Huang and He, 2008; MacDonald et al., 2009) and transcriptional maturation of Paneth cells – a Wnt-dependent cell type – is severely activation of Wnt targets (He et al., 2004; MacDonald and He, 2012; affected. Rab8a knockout crypts show an expansion of Lgr5+ and Tamai et al., 2000; Wehrli et al., 2000). This signaling cascade, which Hopx+ cells in vivo. However, in vitro, the knockout enteroids exhibit is often referred to as the canonical Wnt pathway, plays a fundamental significantly weakened growth that can be partly restored by role in fetal development and adult tissue homeostasis (Clevers, exogenous Wnts or Gsk3β inhibitors. Immunogold labeling and 2006; Clevers and Nusse, 2012). Inappropriate activation of this surface protein isolation identified decreased plasma membrane pathway in diseases, especially colon cancers, has highlighted its localization of Gpr177 in Rab8a knockout Paneth cells and profound influence on cellular behavior (Angers and Moon, 2009; fibroblasts. Upon stimulation by exogenous Wnts, Rab8a-deficient Clevers and Nusse, 2012; de Lau et al., 2007; MacDonald et al., 2009; cells show ligand-induced Lrp6 phosphorylation and transcriptional Nusse et al., 2008; Polakis, 2007; Reya and Clevers, 2005). Certain reporter activation. Rab8a thus controls Wnt delivery in producing Wnt-Fzd complexes activate non-canonical Wnt pathways and cells and is crucial for Paneth cell maturation. Our data highlight the regulate cell migration and polarity via Rho subfamily small profound tissue plasticity that occurs in response to stress induced by GTPases (Boutros and Mlodzik, 1999; Eaton et al., 1996; Fanto depletion of a stem cell niche signal. et al., 2000; Habas et al., 2003, 2001; Sakamori et al., 2014; Strutt et al., 1997; Wallingford and Habas, 2005). KEY WORDS: Rab8a, Gpr177, Wntless, Wnt secretion, Intestinal In Wnt-producing cells, newly synthesized Wnt proteins are lipid stem cell, Paneth cell modified in the endoplasmic reticulum (ER) by an acyltransferase, Porcupine (Takada et al., 2006), and transported by the multi-pass INTRODUCTION transmembrane protein Wntless [also known as G protein-coupled Wnts are cysteine-rich glycolipoproteins that act as paracrine or receptor 177 (Gpr177) in mammals] for exocytosis (Bänziger et al., autocrine ligands believed to engage in short-range signaling (Willert 2006; Bartscherer et al., 2006). Global or tissue-specific ablation of et al., 2003; Willert and Nusse, 2012). Signal transduction in the Wnt- Wntless/Gpr177 in various animals causes phenotypes that responding cell is initiated by binding of Wnts to their seven-pass resemble loss of Wnts (Bänziger et al., 2006; Bartscherer et al., transmembrane Frizzled (Fzd) receptors (Schulte, 2010; Schulte and 2006; Fu et al., 2011), leading to the current notion that Wntless/ Bryja, 2007; Wu and Nusse, 2002). The Wnt-Fzd complex recruits Gpr177 represents the specific and possibly sole transporter for secretion of most Wnts (Ching and Nusse, 2006; Das et al., 2012; Port and Basler, 2010). After Wnt release at the cell surface, Gpr177 1Department of Biological Sciences, Rutgers University, Newark, NJ 07102, USA. is internalized from the plasma membrane via a Clathrin-dependent 2Department of Genetics, Human Genetics Institute of New Jersey, Rutgers University, Piscataway, NJ 08854, USA. 3Department of Biomedical Genetics, pathway to endosomes (Gasnereau et al., 2011; Pan et al., 2008), Center for Oral Biology, James P. Wilmot Cancer Center, Stem Cell and where it is retrieved in a retromer Vps35- and Snx3-dependent Regenerative Medicine Institute, University of Rochester Medical Center, fashion to the Golgi for new rounds of Wnt transport (Belenkaya Rochester, NY 14642, USA. 4Department of Cell Biology, Graduate School of Medicine, Osaka University 2-2, Yamadaoka, Suita, Osaka 565-0871, Japan. et al., 2008; Franch-Marro et al., 2008; Harterink et al., 2011; Port 5Rutgers Cancer Institute of New Jersey, New Brunswick, NJ 08901, USA. et al., 2008; Rojas et al., 2008; Yang et al., 2008). In contrast to the intensive study of retrograde traffic of Gpr177, the cellular *Author for correspondence ([email protected]) machineries that govern anterograde Gpr177-Wnt transport have This is an Open Access article distributed under the terms of the Creative Commons Attribution not been fully explored. It is unclear whether Gpr177-Wnt follows a License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. vesicular membrane bulk flow (Hong and Tang, 1993) or if Wnt exocytosis is subject to regulation by specific secretory machinery Received 16 December 2014; Accepted 16 April 2015 (Das et al., 2012). DEVELOPMENT 2147 RESEARCH ARTICLE Development (2015) 142, 2147-2162 doi:10.1242/dev.121046 Among various cell types that produce distinct cohorts of Wnts et al., 2007). However, the contribution of Rab8 vesicles to surrounding the intestinal stem cell niche (Gregorieff et al., 2005), intestinal crypt homeostasis is not defined. Paneth cells are the major epithelial Wnt producers, expressing A recent screening for Rab modulators of the Wnt pathway Wnt3, Wnt6 and Wnt9b, and co-occupy the crypt bottom with stem identified RAB8B, but not RAB8A, as a crucial regulator of cells. The self-renewal of fast-cycling Lgr5+ stem cells (Barker canonical Wnt signaling in receiving cells by directly interacting et al., 2007), as shown by organoid-forming capacity in culture, is with LRP6 and CK1γ (Demir et al., 2013). We provide evidence enhanced by close association with Paneth cells or by addition of here that, in Wnt-producing cells, Rab8a regulates Gpr177 exogenous Wnt ligands (Sato et al., 2011). The ablation of Paneth anterograde traffic and Wnt secretion. Using immunogold cells in several mouse models caused recoverable loss of Lgr5+ stem labeling of endogenous Gpr177 in native Wnt producers, Wnt cells (Sato et al., 2011). However, Atoh1-deficient mouse intestines secretion and reporter assays, we demonstrate that Rab8a ablation with an absence of Paneth cell differentiation preserved functional impairs Gpr177 trafficking in Wnt producers, attenuating Wnt intestinal epithelia (Durand et al., 2012; Kim et al., 2012), hinting at secretion and canonical Wnt signaling in vivo and ex vivo. Rab8a a high plasticity of crypt cells (Tetteh et al., 2014). In addition to knockout intestinal crypts showed altered cell organization in Paneth cells, subepithelial stromal cells express Wnt2b, Wnt4 and response to decreased extracellular Wnts in the niche. These data Wnt5a (Farin et al., 2012; Gregorieff et al., 2005; Miyoshi et al., shed light on intestinal crypt plasticity in response to stress induced 2012). Wnt5a+ mesothelial cells contribute to regenerating nascent by defective niche signal traffic. crypts after tissue injury (Miyoshi et al., 2012). Intestinal epithelia- specific ablation of Wnt3 (Farin et al., 2012), or Porcupine deletion RESULTS in both epithelia and myofibroblasts (Kabiri et al., 2014; San Roman Gpr177 traffics through Rab8a vesicles et al., 2014), did not cause detectable tissue damage. In culture, We established a stable Henrietta Lacks (HeLa) human cell line Wnt3-deficient intestinal organoids fail to propagate, but expressing 3×Flag-GPR177 to identify regulators for Wnt-GPR177 administration of Wnts was able to restore the growth (Farin et al., trafficking. Using cell lysates extracted in the presence of 1% Triton 2012), collectively suggesting that multiple sources of Wnts X-100, we performed co-immunoprecipitation analyses to identify redundantly support the stem cell niche. potential interactions
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